Method for producing a colorimetric resonant reflection biosensor on rigid surfaces

ABSTRACT

The invention provides a methods for fabricating colorimetric resonant reflection biosensors.

PRIORITY

This application is a continuation-in-part of U.S. application Ser. No.10/180,374, entitled “Method and Instrument for Detecting BiomolecularInteractions” filed Jun. 26, 2002, now U.S. Pat. No. 7,023,544 and U.S.application Ser. No. 10/180,674, entitled “Method and Apparatus forDetecting Biomolecular Interactions” filed Jun. 26, 2002, which arecontinuations-in-part of U.S. application Ser. No. 10/059,060, filedJan. 28, 2002 and U.S. application Ser. No. 10/058,626, filed Jan. 28,2002, now U.S. Pat. No. 6,951,715, which are continuations-in-part ofU.S. application Ser. No. 09/930,352, filed Aug. 15, 2001, which claimsthe benefit of U.S. provisional application 60/244,312 filed Oct. 30,2000; U.S. provisional application 60/283,314 filed Apr. 12, 2001; andU.S. provisional application 60/303,028 filed Jul. 3, 2001, all of whichare incorporated herein in their entirety.

TECHNICAL AREA OF THE INVENTION

This invention provides methods of producing colorimetric resonantreflection biosensors.

BACKGROUND OF THE INVENTION

Colorimetric resonant reflection biosensors have been fabricated onrigid surfaces, such as glass, using photoresist etch techniques. Forexample, a high refractive index dielectric thin film is deposited ontoa glass substrate. A layer of photoresist is deposited over thedielectric thin film. The photoresist is exposed to light so as toselectively expose regions where the dielectric thin film is to beremoved. The glass is immersed into a photoresist chemical developersolution that removes photoresist only from regions where thephotoresist was exposed to higher intensity light. The glass substrateis exposed to an etchant (either a wet chemical solution or a dryreactive ion etch process) which removes the dielectric thin materialfrom areas where the photoresist has been developed away. Any remainingphotoresist is selectively removed. This glass substrate fabricationprocess is costly because each individual biosensor must undergo theexpensive photolithography and etching procedures.

Another method of producing a colorimetric resonant reflection biosensorcomprises spreading a thin layer of epoxy between a rigid “master”template that contains a negative of the desired colorimetric resonantreflection biosensor surface structure onto a flexible sheet of plasticsubstrate such as polycarbonate or polyester. The epoxy is cured and theplastic sheet with cured epoxy is peeled away from the master template.A high refractive index dielectric material is deposited over thestructured plastic sheet. The limitation of the plastic substratefabrication process is that the “cure and peel” procedure for generatinga surface structure replicated within epoxy requires the substrate to beflexible in order to peel the cured structure away from the rigid master(typically implemented as a silicon or glass wafer or a metal film).

Methods of fabricating colorimetric resonant reflection biosensors thatavoid expensive photolithography procedures or that do not require theuse of flexible substrate materials are needed in the art.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a method for fabrication of acolorimetric resonant reflection biosensor structure. A colorimetricresonant reflection biosensor structure comprises a substrate and agrating comprised of, or coated with, a high refractive index dielectricfilm or reflective material. A colorimetric resonant reflectionbiosensor further comprises one or more immobilized specific bindingsubstances on the high refractive index dielectric film. The methodcomprises applying a liquid or semi-solid material that is capable ofbeing transformed or cured into a flexible solid over a rigid masterstructure; transforming the liquid or semi-solid material into aflexible master structure, wherein the flexible master structure has therigid master structure embossed into a first surface of the flexiblemaster structure; peeling the flexible master structure from the rigidmaster structure; placing the first surface of the flexible masterstructure onto a liquid or semisolid layer, wherein the liquid orsemi-solid layer is on a rigid substrate; transforming or curing theliquid or semi-solid layer into a solid layer; peeling the flexiblemaster structure from the solid layer; and applying a high refractiveindex dielectric film or reflective material over the solid layer,whereby a colorimetric resonant reflection biosensor structure isfabricated.

One or more specific binding substances can be immobilized to the highrefractive index film or reflective material of the colorimetricresonant reflection biosensor structure to fabricate a colorimetricresonant reflection biosensor.

The rigid master structure can be etched into the surface of a siliconor glass wafer. The liquid or semi-solid layer can be epoxy, a polymer,a cement, a solvent free radiation addition polymerizable crosslinkablematerial or a resin. The crosslinkable material can be an acrylate epoxyurethane based material. The liquid or semi-solid material can bepolydimethylsiloxane.

An adhesion-enhancing thin film is placed between the liquid orsemi-solid layer and the rigid substrate before the first surface of theflexible master structure is placed on the liquid or semisolid layer.The rigid substrate can be glass, plastic, polymer or epoxy. The liquidor semi-solid layer can be transformed or cured by an electron beam,ultraviolet light or heat.

The flexible master structure can comprise a grating pattern selectedfrom the group consisting of squares, triangles, sinusoidal waves,inverted “u” shapes, lines, circles, ellipses, trapezoids, ovals,rectangles, hexagons, phase-quantized terraced surface relief patternswhose groove pattern resembles a stepped pyramid, and concentric rings.The flexible master structure can comprise a grating pattern having aperiodic spacing of between about 0.1 microns to about 2.0 microns. Theflexible master structure can comprise a submicron grating pattern andcan have a periodic spacing of between about 0.2 microns to about 0.6microns.

Another embodiment of the invention provides a method of producing aflexible master structure for use in fabricating a colorimetric resonantreflection biosensor. The method comprises applying a liquid orsemi-solid material that is capable of being transformed or cured into aflexible master structure over a rigid colorimetric resonant reflectionbiosensor master structure; transforming or curing the liquid orsemi-solid material into a flexible master structure, wherein theflexible master structure has the rigid colorimetric resonant reflectionbiosensor master structure embossed into a first surface of the flexiblemaster structure; and peeling the flexible master structure from therigid colorimetric resonant reflection biosensor master structure;whereby a flexible master structure for use in fabricating acolorimetric resonant reflection biosensor is produced.

Even anther embodiment of the invention provides a method forfabrication of a colorimetric resonant reflection biosensor structure.The method comprises placing a colorimetric resonant reflectionbiosensor flexible master structure onto a liquid or semi-solid layer,wherein the liquid or semi-solid layer is on a rigid substrate, whereinthe flexible master structure is pressed into the liquid or semi-solidlayer; transforming or curing the liquid or semi-solid layer into asolid layer; peeling the flexible colorimetric resonant reflectionbiosensor master structure from the solid layer; and applying a highrefractive index dielectric film or reflective material over the solidlayer, whereby a colorimetric resonant reflection biosensor structure isfabricated.

The method can further comprise the step of immobilizing one or morespecific binding substances to the high refractive index dielectric filmor reflective material, wherein a colorimetric resonant reflectionbiosensor is fabricated.

The invention therefore provides low cost methods of fabricatingcolorimetric resonant reflection biosensors on rigid surfaces.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates a liquid or semi-solid layer, such as PDMS(polydimethylsiloxane), that has been poured over a rigid masterstructure which has been etched into a solid surface.

FIG. 2 demonstrates one embodiment of the invention wherein a flexiblemaster structure is pressed into epoxy to generate a uniformly thinlayer of epoxy between a substrate and a rigid master structure.

FIG. 3 demonstrates one embodiment of a structure of a colorimetricresonant reflection sensor fabricated using the methods of theinvention.

FIG. 4 shows the results when a colorimetric resonant reflectionbiosensor structure fabricated as described in Example 1 was illuminatedwith collimated white light. The expected resonant reflection peakspectra were obtained in air, water, and glycerol.

FIG. 5A shows a cross-sectional view of a biosensor wherein light isshown as illuminating the bottom of the biosensor; however, light canilluminate the biosensor from either the top or the bottom. FIG. 5Bshows a diagram of a biosensor wherein light is shown as illuminatingthe bottom of the biosensor; however, light can illuminate the biosensorfrom either the top or the bottom;

FIGS. 6A–B shows a grating comprising a rectangular grid of squares(FIG. 6A) or holes (FIG. 6B);

FIG. 7 shows a biosensor cross-section profile utilizing a sinusoidallyvarying grating profile;

FIG. 8 shows three types of surface activation chemistry (Amine,Aldehyde, and Nickel) with corresponding chemical linker molecules thatcan be used to covalently attach various types of biomolecule receptorsto a biosensor;

FIG. 9 shows an example of a biosensor used as a microarray;

FIGS. 10A–B shows two biosensor formats that can incorporate acolorimetric resonant reflectance biosensor. FIG. 10A shows a biosensorthat is incorporated into a microtitre plate. FIG. 10B shows a biosensorin a microarray slide format;

FIG. 11 shows an array of arrays concept for using a biosensor platformto perform assays with higher density and throughput;

FIG. 12 demonstrates an example of a biosensor that occurs on the tip ofa fiber probe for in vivo detection of biochemical substances;

FIG. 13 shows an example of the use of two coupled fibers to illuminateand collect reflected light from a biosensor;

FIG. 14 shows resonance wavelength of a biosensor as a function ofincident angle of detection beam;

FIG. 15 shows an example of the use of a beam splitter to enableilluminating and reflected light to share a common collimated opticalpath to a biosensor;

FIG. 16 shows an example of a system for angular scanning of abiosensor;

FIG. 17 shows a colorimetric resonant reflection or transmission filterstructure consisting of a set of concentric rings;

FIG. 18 shows a grid structure comprising a hexagonal grid of holes (ora hexagonal grid of posts) that closely approximates the concentriccircle structure of FIG. 17 without requiring the illumination beam tobe centered upon any particular location of the grid.

FIG. 19 shows a schematic diagram of a detection system.

FIG. 20 shows a graphic representation of how adsorbed material, such asa protein monolayer, will increase the reflected wavelength of abiosensor that comprises a three-dimensional grating.

FIG. 21 shows an embodiment of a colorimetric resonant reflectionbiosensor comprising a one-dimensional grating made according to themethods and compositions of the invention further comprising an optionalcover layer.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for making replicated colorimetricresonant reflection biosensor structures on rigid substrate surfaceswithout the use of photolithography methods or flexible substrates. Themethods facilitate low cost manufacturing of colorimetric resonantreflection biosensors into disposable laboratory items made of, forexample, glass and plastic, such as microscope slides, microarrayslides, petri dishes, and cell culture dishes. For several importantapplications of colorimetric resonant reflection biosensor technology,such as microarray slides, the most common laboratory format that istypically used with laboratory infrastructure (such as liquid handlingdispensers, microarray spotters, incubators, mixers, etc.) involves arigid substrate. Examples include glass microscope slides, petri dishes,and cell culture plates.

Because these laboratory formats are low cost disposable items, amanufacturing process for embedding colorimetric resonant reflectionbiosensors into them must also be performed with low cost. The processdescribed in the invention uses an inexpensive flexible cast taken froma master structure to transfer, for example, a sub-micron structuredsurface to a rigid surface, such as a sheet of glass or plastic.

Methods of Making a Colorimetric Resonant Reflection Biosensor

In general, methods of the invention involve two separate processes. Thefirst process creates a flexible master structure from a rigid masterstructure. The second process utilizes the flexible master structure tocreate a colorimetric resonant reflection biosensor structure on a rigidsubstrate surface.

In one embodiment of the invention, a sub-wavelength surface (SWS)grating is etched into the surface of a rigid master structure such assilicon or glass wafer. Alternatively, a grating can be produced inphotoresist. The nonlinear etching characteristics of photoresist areused to develop the exposed film to create a grating pattern. A rigidmaster structure is then produced from the photoresist. For example, athin silver film is deposited over the photoresist structure to form aconducting layer upon which a thick film of nickel can be electroplatedto form a nickel master plate. The rigid master structure can be formedso as to produce a flexible master structure comprising a gratingpattern selected from the group consisting of squares, triangles,sinusoidal waves, inverted “u” shapes, lines, circles, ellipses,trapezoids, ovals, rectangles, hexagons, phase-quantized terracedsurface relief patterns whose groove pattern resembles a stepped pyramidand concentric rings. A grating can be a one-, two- or three-dimensionalgrating. Preferably, the grating is a sub-wavelength structured surfacethat is capable of functioning as a colorimetric resonant reflectionbiosensor. Preferably, the flexible master structure comprises a gratingpattern having a periodic spacing of between about 0.1 microns to about2.0 microns. Even more preferably, the flexible master structurecomprises a submicron grating pattern, that is, the periodic spacing isless than 1 micron. In one embodiment the periodic spacing is betweenabout 0.2 microns to about 0.6 microns. In another embodiment, theperiodic spacing is between about 0.01 microns and about 0.9 microns.

A liquid or semi-solid material that is capable of being transformed toa flexible solid is applied or poured over the rigid master structure sothat it conforms to the contours of the rigid master structure surface.Liquid or semi-solid materials include, for example, thermoplasticelastomers such as polydimethylsiloxane (PDMS). The liquid or semi-solidmaterial is cured or transformed by, for example heat, into a flexiblesolid to form a flexible master structure. The flexible master structurehas the rigid master structure embossed into a first surface of theflexible master structure. For example, PDMS can be cured into aflexible solid using heat. The cured or transformed flexible mastersolid structure is peeled away from the rigid master structure.

A liquid or semi-solid layer material that is capable of beingtransformed into a solid is applied to a rigid substrate such as glass,plastic, polymer or epoxy to form a liquid or semi-solid material layer.The liquid or semi-solid layer material can be, for example, epoxy, apolymer, a cement, a solvent free radiation addition polymerizable crosslinkable material, such as an acrylate epoxy urethane based material,and a resin. An adhesion-enhancing thin film can be applied to the rigidsubstrate before the liquid or semi-solid layer material is applied tothe substrate. Adhesion-enhancing films can comprise, for example,hexamethyldisilane (HMDS).

A flexible master structure has the rigid master structure embossed intoa first surface. The first surface of the flexible master structure isapplied over the liquid or semi-solid layer material. The flexiblemaster structure is pressed into the liquid or semi-solid layermaterial, using for example, a roller, to generate a uniformly thinlayer of the liquid or semi-solid layer material between the substrateand flexible master structure. The liquid or semi-solid layer materialis transformed or cured into a solid layer. The layer material can becured or transformed by for example, beat, an electron beam, orultraviolet light. For example, an epoxy layer can be cured using light.The flexible master structure is peeled away from the solid layer. Ahigh refractive index dielectric thin film can be deposited over thetransformed or cured solid layer, by, for example, sputter deposition.Specific binding substances can be immobilized on the surface of thehigh refractive index dielectric thin film to form a colorimetricresonant reflection biosensor.

Colorimetric Resonant Reflection Biosensors

A colorimetric resonant reflection biosensor comprises a subwavelengthstructured surface (SWS), which is used to create a sharp opticalresonant reflection at a particular wavelength that can be used to, forexample, track with high sensitivity the interaction of biologicalmaterials, such as specific binding substances or binding partners orboth. See e.g., U.S. application Ser. Nos. 09/930,352; 10/059,060, and10/058,626, all of which are incorporated herein in their entirety.

The microreplicating methods of the invention can be used to make asubwavelength structured surface (SWS), such as a colorimetric resonantreflection diffractive grating surface. See U.S. application Ser. Nos.10/058,626 and 10/059,060. Such a grating surface can be used to createa sharp optical resonant reflection at a particular wavelength that canbe used to track with high sensitivity the interaction of biologicalmaterials, such as specific binding substances or binding partners orboth. The colorimetric resonant reflection diffractive grating surfaceacts as a surface binding platform for specific binding substances.

Subwavelength structured surfaces are an unconventional type ofdiffractive optic that can mimic the effect of thin-film coatings. (Peng& Morris, “Resonant scattering from two-dimensional gratings,” J. Opt.Soc. Am. A, Vol. 13, No. 5, p. 993, May; Magnusson, & Wang, “Newprinciple for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022,August, 1992; Peng & Morris, “Experimental demonstration of resonantanomalies in diffraction from two-dimensional gratings,” Optics Letters,Vol. 21, No. 8, p. 549, April, 1996). A SWS structure contains a one-,two- or three-dimensional grating in which the grating period is smallcompared to the wavelength of incident light so that no diffractiveorders other than the reflected and transmitted zeroth orders areallowed to propagate. A SWS structure can comprise an optical gratingsandwiched between a substrate layer and a cover layer that fills thegrating. Optionally, a cover layer is not used. When the effective indexof refraction of the grating region is greater than the substrate or thecover layer, a waveguide is created. When a filter is designed properly,incident light passes into the waveguide region and propagates as aleaky mode. An optical grating structure selectively couples light at anarrow band of wavelengths into the waveguide. The light propagates onlya very short distance (on the order of 10–100 micrometers), undergoesscattering, and couples with the forward- and backward-propagatingzeroth-order light. This highly sensitive coupling condition can producea resonant grating effect on the reflected radiation spectrum, resultingin a narrow band of reflected or transmitted wavelengths. The depth andperiod of the one-, two- or three-dimensional grating are less than thewavelength of the resonant grating effect.

The reflected or transmitted wavelengths produced by this structure canbe modulated by the addition of molecules such as specific bindingsubstances or binding partners or both to the upper surface the gratingsurface or cover layer. The added molecules increase the optical pathlength of incident radiation through the structure, and thus modify thewavelength at which maximum reflectance or transmittance will occur.

In one embodiment, a biosensor, when illuminated with white light, isdesigned to reflect only a single wavelength or a narrow band ofwavelengths. When specific binding substances are attached to thesurface of the biosensor, the reflected wavelength is shifted due to thechange of the optical path of light that is coupled into the grating. Bylinking specific binding substances to a biosensor surface,complementary binding partner molecules can be detected without the useof any kind of fluorescent probe or particle label. The detectiontechnique is capable of resolving changes of, for example, ˜0.1 nmthickness of protein binding, and can be performed with the biosensorsurface either immersed in fluid or dried.

A detection system can include, for example, a light source thatilluminates a small spot of a biosensor at normal incidence through, forexample, a fiber optic probe, and a spectrometer that collects thereflected light through, for example, a second fiber optic probe also atnormal incidence. Because no physical contact occurs between theexcitation/detection system and the biosensor surface, no specialcoupling prisms are required and the biosensor can be easily adapted toany commonly used assay platform including, for example, microtiterplates and microarray slides. A single spectrometer reading can beperformed in several milliseconds; it is thus possible to quicklymeasure a large number of molecular interactions taking place inparallel upon a biosensor surface, and to monitor reaction kinetics inreal time.

This technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels would alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by the compositionsand methods of the invention.

FIGS. 5A and 5B are diagrams of an example of a colorimetric resonantreflection diffractive grating biosensor. In FIG. 5, n₁ represents anoptical grating. n₂ represents the high refractive index layer. Layerthicknesses (i.e. an optional cover layer, one or more specific bindingsubstances, or an optical grating) are selected to achieve resonantwavelength sensitivity to additional molecules on the top surface. Thegrating period is selected to achieve resonance at a desired wavelength.

A SWS biosensor comprises an optical grating, a substrate layer thatsupports the grating, and one or more specific binding substancesimmobilized on the surface of the grating opposite of the substratelayer. Optionally, a cover layer covers the grating surface. An opticalgrating made according to the invention is coated with a high refractiveindex dielectric film which can be comprised of a material thatincludes, for example, zinc sulfide, titanium dioxide, tantalum oxide,and silicon nitride. A cross-sectional profile of a grating with opticalfeatures can comprise any periodically repeating function, for example,a “square-wave.” An optical grating can also comprise a repeatingpattern of shapes selected from the group consisting of lines, squares,circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals,rectangles, and hexagons.

Sensor Characteristics

Linear gratings (i.e., one dimensional gratings) have resonantcharacteristics where the illuminating light polarization is orientedperpendicular to the grating period. A schematic diagram of a lineargrating structure is shown in FIG. 21. A colorimetric resonantreflection biosensor can also comprise, for example, a two-dimensionalgrating, e.g., a hexagonal array of holes (see FIG. 6B) or squares (seeFIG. 6A). Other shapes can be used as well. A linear grating has thesame pitch (i.e. distance between regions of high and low refractiveindex), period, layer thicknesses, and material properties as ahexagonal array grating. However, light must be polarized perpendicularto the grating lines in order to be resonantly coupled into the opticalstructure. Therefore, a polarizing filter oriented with its polarizationaxis perpendicular to the linear grating must be inserted between theillumination source and the biosensor surface. Because only a smallportion of the illuminating light source is correctly polarized, alonger integration time is required to collect an equivalent amount ofresonantly reflected light compared to a hexagonal grating.

An optical grating can also comprise, for example, a “stepped” profile,in which high refractive index regions of a single, fixed height areembedded within a lower refractive index cover layer. The alternatingregions of high and low refractive index provide an optical waveguideparallel to the top surface of the biosensor.

It is also possible to make a resonant biosensor in which the highrefractive index material is not stepped, but which varies with lateralposition. FIG. 7 shows a profile in which the high refractive indexmaterial which coats a two-dimensional grating, n₁, is sinusoidallyvarying in height. n₂ represents an optional cover layer, and t₂represents the thickness of the grating. To produce a resonantreflection at a particular wavelength, the period of the sinusoid isidentical to the period of an equivalent stepped structure. The resonantoperation of the sinusoidally varying structure and its functionality asa biosensor has been verified using GSOLVER (Grating Solver DevelopmentCompany, Allen, Tex., USA) computer models.

A biosensor of the invention can further comprise a cover layer on thesurface of an optical grating opposite of a substrate layer. Where acover layer is present, the one or more specific binding substances areimmobilized on the surface of the cover layer opposite of the grating.Preferably, a cover layer comprises a material that has a lowerrefractive index than a material that comprises the grating. A coverlayer can be comprised of, for example, glass (including spin-on glass(SOG)), epoxy, or plastic.

For example, various polymers that meet the refractive index requirementof a biosensor can be used for a cover layer. SOG can be used due to itsfavorable refractive index, ease of handling, and readiness of beingactivated with specific binding substances using the wealth of glasssurface activation techniques. When the flatness of the biosensorsurface is not an issue for a particular system setup, a gratingstructure of SiN/glass can directly be used as the sensing surface, theactivation of which can be done using the same means as on a glasssurface.

Resonant reflection can also be obtained without a planarizing coverlayer over an optical grating. For example, a biosensor can contain onlya substrate coated with a structured thin film layer of high refractiveindex material. Without the use of a planarizing cover layer, thesurrounding medium (such as air or water) fills the grating. Therefore,specific binding substances are immobilized to the biosensor on allsurfaces of an optical grating exposed to the specific bindingsubstances, rather than only on an upper surface.

In general, a biosensor of the invention will be illuminated with whitelight that will contain light of every polarization angle. Theorientation of the polarization angle with respect to repeating featuresin a biosensor grating will determine the resonance wavelength. Forexample, a “linear grating” (i.e., a one-dimensional grating) biosensorconsisting of a set of repeating lines and spaces will have two opticalpolarizations that can generate separate resonant reflections. Lightthat is polarized perpendicularly to the lines is called “s-polarized,”while light that is polarized parallel to the lines is called“p-polarized.” Both the s and p components of incident light existsimultaneously in an unfiltered illumination beam, and each generates aseparate resonant signal. A biosensor can generally be designed tooptimize the properties of only one polarization (the s-polarization),and the non-optimized polarization is easily removed by a polarizingfilter.

In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate biosensor structure can be used that consists of a set ofconcentric rings. In this structure, the difference between the insidediameter and the outside diameter of each concentric ring is equal toabout one-half of a grating period. Each successive ring has an insidediameter that is about one grating period greater than the insidediameter of the previous ring. The concentric ring pattern extends tocover a single sensor location—such as a microarray spot or a microtiterplate well. Each separate microarray spot or microtiter plate well has aseparate concentric ring pattern centered within it. See, e.g., FIG. 17.All polarization directions of such a structure have the samecross-sectional profile. The concentric ring structure must beilluminated precisely on-center to preserve polarization independence.The grating period of a concentric ring structure is less than thewavelength of the resonantly reflected light. The grating period isabout 0.01 micron to about 1 micron. The grating depth is about 0.01 toabout 1 micron.

In another embodiment, an array of holes or posts are arranged toclosely approximate the concentric circle structure described abovewithout requiring the illumination beam to be centered upon anyparticular location of the grid. See e.g. FIG. 18. Such an array patternis automatically generated by the optical interference of three laserbeams incident on a surface from three directions at equal angles. Inthis pattern, the holes (or posts) are centered upon the corners of anarray of closely packed hexagons as shown in FIG. 18. The holes or postsalso occur in the center of each hexagon. Such a hexagonal grid of holesor posts has three polarization directions that “see” the samecross-sectional profile. The hexagonal grid structure, therefore,provides equivalent resonant reflection spectra using light of anypolarization angle. Thus, no polarizing filter is required to removeunwanted reflected signal components. The period of the holes or postscan be about 0.01 microns to about 1 micron and the depth or height canbe about 0.01 microns to about 1 micron.

Another grating that can be produced using the methods of the inventionis a volume surface-relief volume diffractive grating (a SRVD grating),also referred to as a three-dimensional grating. SRVD gratings have asurface that reflects predominantly at a particular narrow band ofoptical wavelengths when illuminated with a broad band of opticalwavelengths. Where specific binding substances and/or binding partnersare immobilized on a SRVD grating, producing a SRVD biosensor, thereflected narrow band of wavelengths of light is shifted.One-dimensional surfaces, such as thin film interference filters andBragg reflectors, can select a narrow range of reflected or transmittedwavelengths from a broadband excitation source, however, the depositionof additional material, such as specific binding substances and/orbinding partners onto their upper surface results only in a change inthe resonance linewidth, rather than the resonance wavelength. Incontrast, SRVD biosensors have the ability to alter the reflectedwavelength with the addition of material, such as specific bindingsubstances and/or binding partners to the surface. The depth and periodof relief volume diffraction structures are less than the resonancewavelength of light reflected from a biosensor.

A three-dimensional surface-relief volume diffractive grating can be,for example, a three-dimensional phase-quantized terraced surface reliefpattern whose groove pattern resembles a stepped pyramid. When such agrating is illuminated by a beam of broadband radiation, light will becoherently reflected from the equally spaced terraces at a wavelengthgiven by twice the step spacing times the index of refraction of thesurrounding medium. Light of a given wavelength is resonantly diffractedor reflected from the steps that are a half-wavelength apart, and with abandwidth that is inversely proportional to the number of steps. Thereflected or diffracted color can be controlled by the deposition of ahigh refractive index layer so that a new wavelength is selected,depending on the index of refraction of the coating. As an example, athin-film layer of zinc sulfide, titanium dioxide, tantalum oxide, orsilicon nitride can be sputter deposited onto the grating.

An example of a three-dimensional phase-quantized terraced surfacerelief pattern is a pattern that resembles a stepped pyramid. Eachinverted pyramid is approximately 1 micron in diameter, preferably, eachinverted pyramid can be about 0.5 to about 5 microns diameter, includingfor example, about 1 micron. The pyramid structures can be close-packedso that a typical microarray spot with a diameter of about 150–200microns can incorporate several hundred stepped pyramid structures. Therelief volume diffraction structures have a period of about 0.1 to about1 micron and a depth of about 0.1 to about 1 micron. FIG. 20demonstrates how individual microarray locations (with an entiremicroarray spot incorporating hundreds of pyramids now represented by asingle pyramid for one microarray spot) can be optically queried todetermine if specific binding substances or binding partners areadsorbed onto the surface. When the biosensor is illuminated with whitelight, pyramid structures without significant bound material willreflect wavelengths determined by the step height of the pyramidstructure. When higher refractive index material, such as bindingpartners or specific binding substances, are incorporated over thereflective metal surface, the reflected wavelength is modified to shifttoward longer wavelengths. The color that is reflected from the terracedstep structure is theoretically given as twice the step height times theindex of refraction of a reflective material that is coated onto thefirst surface of a sheet material of a SRVD biosensor. A reflectivematerial can be, for example silver, aluminum, or gold.

One or more specific binding substances, as described above, areimmobilized on the reflective material of a SRVD biosensor. One or morespecific binding substances can be arranged in microarray of distinctlocations, as described above, on the reflective material.

Because the reflected wavelength of light from a SRVD biosensor isconfined to a narrow bandwidth, very small changes in the opticalcharacteristics of the surface manifest themselves in easily observedchanges in reflected wavelength spectra. The narrow reflection bandwidthprovides a surface adsorption sensitivity advantage compared toreflectance spectrometry on a flat surface.

A SRVD biosensor reflects light predominantly at a first single opticalwavelength when illuminated with a broad band of optical wavelengths,and reflects light at a second single optical wavelength when one ormore specific binding substances are immobilized on the reflectivesurface. The reflection at the second optical wavelength results fromoptical interference. A SRVD biosensor also reflects light at a thirdsingle optical wavelength when the one or more specific bindingsubstances are bound to their respective binding partners, due tooptical interference.

Readout of the reflected color can be performed serially by focusing amicroscope objective onto individual microarray spots and reading thereflected spectrum, or in parallel by, for example, projecting thereflected image of the microarray onto a high resolution color CCDcamera.

In one embodiment of the invention, an optical device is provided. Anoptical device comprises a structure similar to a biosensor of theinvention; however, an optical device does not comprise one of morebinding substances immobilized on the grating. An optical device can beused as, for example, a narrow band optical filter.

Specific Binding Substances and Binding Partners

One or more specific binding substances can be immobilized oncolorimetric resonant reflectance gratings produced by the methods ofthe invention by for example, physical adsorption or by chemical bindingwhere a specific binding substance is bound to a colorimetric resonantreflectance grating, to produce a biosensor. A specific bindingsubstance can be, for example, a nucleic acid, polypeptide, antigen,polyclonal antibody, monoclonal antibody, single chain antibody (scFv),F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule,cell, virus, bacteria, or biological sample. A biological sample can befor example, blood, plasma, serum, gastrointestinal secretions,homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum,cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lunglavage fluid, semen, lymphatic fluid, tears, or prostatitc fluid.

Preferably, one or more specific binding substances are arranged in amicroarray of distinct locations on a biosensor. A microarray ofspecific binding substances comprises one or more specific bindingsubstances on a surface of a biosensor such that a surface contains manydistinct locations, each with a different specific binding substance orwith a different amount of a specific binding substance. For example, anarray can comprise 1, 10, 100, 1,000, 10,000, or 100,000 distinctlocations. Such a biosensor surface is called a microarray because oneor more specific binding substances are typically laid out in a regulargrid pattern in x-y coordinates. However, a microarray of the inventioncan comprise one or more specific binding substances laid out in anytype of regular or irregular pattern. For example, distinct locationscan define a microarray of spots of one or more specific bindingsubstances. A microarray spot can be about 50 to about 500 microns indiameter. A microarray spot can also be about 150 to about 200 micronsin diameter. One or more specific binding substances can be bound totheir specific binding partners.

A microarray on a biosensor of the invention can be created by placingmicrodroplets of one or more specific binding substances onto, forexample, an x-y grid of locations on an optical grating or cover layersurface. When the biosensor is exposed to a test sample comprising oneor more binding partners, the binding partners will be preferentiallyattracted to distinct locations on the microarray that comprise specificbinding substances that have high affinity for the binding partners.Some of the distinct locations will gather binding partners onto theirsurface, while other locations will not.

A specific binding substance specifically binds to a binding partnerthat is added to the surface of a biosensor of the invention. A specificbinding substance specifically binds to its binding partner, but doesnot substantially bind other binding partners added to the surface of abiosensor. For example, where the specific binding substance is anantibody and its binding partner is a particular antigen, the antibodyspecifically binds to the particular antigen, but does not substantiallybind other antigens. A binding partner can be, for example, a nucleicacid, polypeptide, antigen, polyclonal antibody, monoclonal antibody,single chain antibody (scFv), F(ab) fragment, F(ab′)₂ fragment, Fvfragment, small organic molecule, cell, virus, bacteria, and biologicalsample. A biological sample can be, for example, blood, plasma, serum,gastrointestinal secretions, homogenates of tissues or tumors, synovialfluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinalfluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid,tears, and prostatitc fluid.

One example of a microarray of the invention is a nucleic acidmicroarray, in which each distinct location within the array contains adifferent nucleic acid molecule. In this embodiment, the spots withinthe nucleic acid microarray detect complementary chemical binding withan opposing strand of a nucleic acid in a test sample.

While microtiter plates are the most common format used for biochemicalassays, microarrays are increasingly seen as a means for maximizing thenumber of biochemical interactions that can be measured at one timewhile minimizing the volume of precious reagents. By application ofspecific binding substances with a microarray spotter onto a biosensorof the invention, specific binding substance densities of 10,000specific binding substances/in² can be obtained. By focusing anillumination beam to interrogate a single microarray location, abiosensor can be used as a label-free microarray readout system. Amicroarray can also be illuminated by a line scanner, wherein an entireline or row of microarray locations are illuminated at once. A CCDcamera can also be used to scan a microarray. A CCD camera comprises anarray of photosensitive elements upon which the microarray image isprojected. The value of each pixel represents the light intensity forthe corresponding area of the microarray. The result of scanning themicroarray is the production of is one or more 2-dimensional images. Theimages are analyzed to determine the extent of binding to themicroarray.

Immobilization of One or More Specific Binding Substances

Immobilization of one or more binding substances onto a biosensor isperformed so that a specific binding substance will not be washed awayby rinsing procedures, and so that its binding to binding partners in atest sample is unimpeded by the biosensor surface. Several differenttypes of surface chemistry strategies have been implemented for covalentattachment of specific binding substances to, for example, glass for usein various types of microarrays and biosensors. See, e.g., FIG. 8. Thesesame methods can be readily adapted to a biosensor of the invention.Surface preparation of a biosensor so that it contains the correctfunctional groups for binding one or more specific binding substances isan integral part of the biosensor manufacturing process.

One or more specific binding substances can be attached to a biosensorsurface by physical adsorption (i.e., without the use of chemicallinkers) or by chemical binding (i.e., with the use of chemicallinkers). Chemical binding can generate stronger attachment of specificbinding substances on a biosensor surface and provide definedorientation and conformation of the surface-bound molecules.

Liquid-Containing Vessels

A grating of the invention can comprise an inner surface, for example, abottom surface of a liquid-containing vessel. A liquid-containing vesselcan be, for example, a microtiter plate well, a test tube, a petri dish,or a microfluidic channel. One embodiment of this invention is abiosensor that is incorporated into any type of microtiter plate. Forexample, a biosensor can be incorporated into the bottom surface of amicrotiter plate by assembling the walls of the reaction vessels overthe resonant reflection surface, as shown in FIG. 10A and 10B, so thateach reaction “spot” can be exposed to a distinct test sample.Therefore, each individual microtiter plate well can act as a separatereaction vessel. Separate chemical reactions can, therefore, occurwithin adjacent wells without intermixing reaction fluids and chemicallydistinct test solutions can be applied to individual wells.

Several methods for attaching a biosensor or grating of the invention tothe bottom surface of bottomless microtiter plates can be used,including, for example, adhesive attachment, ultrasonic welding, andlaser welding.

The most common assay formats for pharmaceutical high-throughputscreening laboratories, molecular biology research laboratories, anddiagnostic assay laboratories are microtiter plates. The plates arestandard-sized plastic cartridges that can contain 96, 384, or 1536individual reaction vessels arranged in a grid. Due to the standardmechanical configuration of these plates, liquid dispensing, roboticplate handling, and detection systems are designed to work with thiscommon format. A biosensor of the invention can be incorporated into thebottom surface of a standard microtiter plate. See, e g., FIG. 10A.Because the biosensor surface can be fabricated in large areas, andbecause the readout system does not make physical contact with thebiosensor surface, an arbitrary number of individual biosensor areas canbe defined that are only limited by the focus resolution of theillumination optics and the x-y stage that scans theillumination/detection probe across the biosensor surface.

Methods of using Biosensors

Biosensors can be used to study one or a number of specific bindingsubstance/binding partner interactions in parallel. Binding of one ormore specific binding substances to their respective binding partnerscan be detected, without the use of labels, by applying one or morebinding partners to a biosensor that have one or more specific bindingsubstances immobilized on their surfaces. A biosensor is illuminatedwith light and a maxima in reflected wavelength, or a minima intransmitted wavelength of light is detected from the biosensor. If oneor more specific binding substances have bound to their respectivebinding partners, then the reflected wavelength of light is shifted ascompared to a situation where one or more specific binding substanceshave not bound to their respective binding partners. Where a biosensoris coated with an array of distinct locations containing the one or morespecific binding substances, then a maxima in reflected wavelength orminima in transmitted wavelength of light is detected from each distinctlocation of the biosensor.

In one embodiment of the invention, a variety of specific bindingsubstances, for example, antibodies, can be immobilized in an arrayformat onto a biosensor of the invention. See, e.g., FIG. 9. Thebiosensor is then contacted with a test sample of interest comprisingbinding partners, such as proteins. Only the proteins that specificallybind to the antibodies immobilized on the biosensor remain bound to thebiosensor. Such an approach is essentially a large-scale version of anenzyme-linked immunosorbent assay; however, the use of an enzyme orfluorescent label is not required. For high-throughput applications,biosensors can be arranged in an array of arrays, wherein severalbiosensors comprising an array of specific binding substances arearranged in an array. See, e.g., FIG. 11. Such an array of arrays canbe, for example, dipped into microtiter plate to perform many assays atone time. In another embodiment, a biosensor can occur on the tip of afiber probe for in vivo detection of biochemical substance. See, FIG.12.

The activity of an enzyme can be detected by applying one or moreenzymes to a biosensor to which one or more specific binding substanceshave been immobilized. The biosensor is washed and illuminated withlight. The reflected wavelength of light is detected from the biosensor.Where the one or more enzymes have altered the one or more specificbinding substances of the biosensor by enzymatic activity, the reflectedwavelength of light is shifted.

Additionally, a test sample, for example, cell lysates containingbinding partners can be applied to a biosensor of the invention,followed by washing to remove unbound material. The binding partnersthat bind to a biosensor can be eluted from the biosensor and identifiedby, for example, mass spectrometry. Optionally, a phage DNA displaylibrary can be applied to a biosensor of the invention followed bywashing to remove unbound material. Individual phage particles bound tothe biosensor can be isolated and the inserts in these phage particlescan then be sequenced to determine the identity of the binding partner.

For the above applications, and in particular proteomics applications,the ability to selectively bind material, such as binding partners froma test sample onto a biosensor of the invention, followed by the abilityto selectively remove bound material from a distinct location of thebiosensor for further analysis is advantageous. Biosensors of theinvention are also capable of detecting and quantifying the amount of abinding partner from a sample that is bound to a biosensor arraydistinct location by measuring the shift in reflected wavelength oflight. For example, the wavelength shift at one distinct biosensorlocation can be compared to positive and negative controls at otherdistinct biosensor locations to determine the amount of a bindingpartner that is bound to a biosensor array distinct location.

Detection Systems

A detection system can comprise a biosensor a light source that directslight to the biosensor, and a detector that detects light reflected fromthe biosensor. In one embodiment, it is possible to simplify the readoutinstrumentation by the application of a filter so that only positiveresults over a determined threshold trigger a detection.

A light source can illuminate a biosensor from its top surface, i.e.,the surface to which one or more specific binding substances areimmobilized or from its bottom surface. By measuring the shift inresonant wavelength at each distinct location of a biosensor of theinvention, it is possible to determine which distinct locations havebinding partners bound to them. The extent of the shift can be used todetermine the amount of binding partners in a test sample and thechemical affinity between one or more specific binding substances andthe binding partners of the test sample.

A biosensor can be illuminated twice. The first measurement determinesthe reflectance spectra of one or more distinct locations of a biosensorarray with one or more specific binding substances immobilized on thebiosensor. The second measurement determines the reflectance spectraafter one or more binding partners are applied to a biosensor. Thedifference in peak wavelength between these two measurements is ameasurement of the amount of binding partners that have specificallybound to a biosensor or one or more distinct locations of a biosensor.This method of illumination can control for small nonuniformities in asurface of a biosensor that can result in regions with slight variationsin the peak resonant wavelength. This method can also control forvarying concentrations or molecular weights of specific bindingsubstances immobilized on a biosensor.

Computer simulation can be used to determine the expected dependencebetween a peak resonance wavelength and an angle of incidentillumination. The substrate chosen was glass (n_(substratc)=1.50). Thegrating is an optical pattern of silicon nitride squares (t₂=180 nm,n₂=2.01 (n=refractive index)), k₂=0.001 (k=absorption coefficient)) witha period of 510 nm, and a filling factor of 56.2% (i.e., 56.2% of thesurface is covered with silicon nitride squares while the rest is thearea between the squares). The areas between silicon nitride squares arefilled with a lower refractive index material. The same material alsocovers the squares and provides a uniformly flat upper surface. For thissimulation, a glass layer was selected (n₁=1.40) that covers the siliconnitride squares by t₂=100 nm.

The reflected intensity as a function of wavelength was modeled usingGSOLVER software, which utilizes full 3-dimensional vector code usinghybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination can be from any incidence and any polarization.

FIG. 14 plots the dependence of the peak resonant wavelength upon theincident illumination angle. The simulation shows that there is a strongcorrelation between the angle of incident light, and the peak wavelengththat is measured. This result implies that the collimation of theilluminating beam, and the alignment between the illuminating beam andthe reflected beam will directly affect the resonant peak linewidth thatis measured. If the collimation of the illuminating beam is poor, arange illuminating angles will be incident on the biosensor surface, anda wider resonant peak will be measured than if purely collimated lightwere incident.

Because the lower sensitivity limit of a biosensor is related to theability to determine the peak maxima, it is important to measure anarrow resonant peak. Therefore, the use of a collimating illuminationsystem with the biosensor provides for the highest possible sensitivity.

One type of detection system for illuminating the biosensor surface andfor collecting the reflected light is a probe containing, for example,six illuminating optical fibers that are connected to a light source,and a single collecting optical fiber connected to a spectrometer. Thenumber of fibers is not critical, any number of illuminating orcollecting fibers are possible. The fibers are arranged in a bundle sothat the collecting fiber is in the center of the bundle, and issurrounded by the six illuminating fibers. The tip of the fiber bundleis connected to a collimating lens that focuses the illumination ontothe surface of the biosensor.

In this probe arrangement, the illuminating and collecting fibers areside-by-side. Therefore, when the collimating lens is correctly adjustedto focus light onto the biosensor surface, one observes six clearlydefined circular regions of illumination, and a central dark region.Because the biosensor does not scatter light, but rather reflects acollimated beam, no light is incident upon the collecting fiber, and noresonant signal is observed. Only by defocusing the collimating lensuntil the six illumination regions overlap into the central region isany light reflected into the collecting fiber. Because only defocused,slightly uncollimated light can produce a signal, the biosensor is notilluminated with a single angle of incidence, but with a range ofincident angles. The range of incident angles results in a mixture ofresonant wavelengths due to the dependence shown in FIG. 14. Thus, widerresonant peaks are measured than might otherwise be possible.

Therefore, it is desirable for the illuminating and collecting fiberprobes to spatially share the same optical path. Several methods can beused to co-locate the illuminating and collecting optical paths. Forexample, a single illuminating fiber, which is connected at its firstend to a light source that directs light at the biosensor, and a singlecollecting fiber, which is connected at its first end to a detector thatdetects light reflected from the biosensor, can each be connected attheir second ends to a third fiber probe that can act as both anilluminator and a collector. The third fiber probe is oriented at anormal angle of incidence to the biosensor and supportscounter-propagating illuminating and reflecting optical signals. Anexample of such a detection system is shown in FIG. 13.

Another method of detection involves the use of a beam splitter thatenables a single illuminating fiber, which is connected to a lightsource, to be oriented at a 90 degree angle to a collecting fiber, whichis connected to a detector. Light is directed through the illuminatingfiber probe into the beam splitter, which directs light at thebiosensor. The reflected light is directed back into the beam splitter,which directs light into the collecting fiber probe. An example of sucha detection device is shown in FIG. 15. A beam splitter allows theilluminating light and the reflected light to share a common opticalpath between the beam splitter and the biosensor, so perfectlycollimated light can be used without defocusing.

Angular Scanning

Detection systems of the invention are based on collimated white lightillumination of a biosensor surface and optical spectroscopy measurementof the resonance peak of the reflected beam. Molecular binding on thesurface of a biosensor is indicated by a shift in the peak wavelengthvalue, while an increase in the wavelength corresponds to an increase inmolecular absorption.

As shown in theoretical modeling and experimental data, the resonancepeak wavelength is strongly dependent on the incident angle of thedetection light beam. FIG. 14 depicts this dependence as modeled for abiosensor of the invention. Because of the angular dependence of theresonance peak wavelength, the incident white light needs to be wellcollimated. Angular dispersion of the light beam broadens the resonancepeak, and reduces biosensor detection sensitivity. In addition, thesignal quality from the spectroscopic measurement depends on the powerof the light source and the sensitivity of the detector. In order toobtain a high signal-to-noise ratio, an excessively long integrationtime for each detection location can be required, thus lengtheningoverall time to readout a biosensor plate. A tunable laser source can beused for detection of grating resonance, but is expensive.

In one embodiment of the invention, these disadvantages are addressed byusing a laser beam for illumination of a biosensor, and a light detectorfor measurement of reflected beam power. A scanning mirror device can beused for varying the incident angle of the laser beam, and an opticalsystem is used for maintaining collimation of the incident laser beam.See, e.g., “Optical Scanning” (Gerald F. Marchall ed., Marcel Dekker(1991). Any type of laser scanning can be used. For example, a scanningdevice that can generate scan lines at a rate of about 2 lines to about1,000 lines per second is useful in the invention. In one embodiment ofthe invention, a scanning device scans from about 50 lines to about 300lines per second.

In one embodiment, the reflected light beam passes through part of thelaser scanning optical system, and is measured by a single lightdetector. The laser source can be a diode laser with a wavelength of,for example, 780 nm, 785 nm, 810 nm, or 830 nm. Laser diodes such asthese are readily available at power levels up to 150 mW, and theirwavelengths correspond to high sensitivity of Si photodiodes. Thedetector thus can be based on photodiode biosensors. An example of sucha detection system is shown in FIG. 19. A light source (300) provideslight to a scanner device (400), which directs the light into an opticalsystem (500). The optical system (500) directs light to a biosensor(600). Light is reflected from the biosensor (600) to the optical system(500), which then directs the light into a light signal detector (700).One embodiment of a detection system is shown in FIG. 16, whichdemonstrates that while the scanning mirror changes its angularposition, the incident angle of the laser beam on the surface changes bynominally twice the mirror angular displacement. The scanning mirrordevice can be a linear galvanometer, operating at a frequency of about 2Hz up to about 120 Hz, and mechanical scan angle of about 10 degrees toabout 20 degrees. In this example, a single scan can be completed withinabout 10 msec. A resonant galvanometer or a polygon scanner can also beused. The example shown in FIG. 16 includes a simple optical system forangular scanning. It consists of a pair of lenses with a common focalpoint between them. The optical system can be designed to achieveoptimized performance for laser collimation and collection of reflectedlight beam.

The angular resolution depends on the galvanometer specification, andreflected light sampling frequency. Assuming galvanometer resolution of30 arcsec mechanical, corresponding resolution for biosensor angularscan is 60 arcsec, i.e. 0.017 degree. In addition, assume a samplingrate of 100 ksamples/sec, and 20 degrees scan within 10 msec. As aresult, the quantization step is 20 degrees for 1000 samples, i.e. 0.02degree per sample. In this example, a resonance peak width of 0.2degree, as shown by Peng and Morris (Experimental demonstration ofresonant anomalies in diffraction from two-dimensional gratings, OpticsLett., 21:549 (1996)), will be covered by 10 data points, each of whichcorresponds to resolution of the detection system.

The advantages of such a detection system includes: excellentcollimation of incident light by a laser beam, high signal-to-noiseratio due to high beam power of a laser diode, low cost due to a singleelement light detector instead of a spectrometer, and high resolution ofresonance peak due to angular scanning.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

EXAMPLE

A rigid master structure was etched into the surface of a silicon orglass wafer. PDMS (Polydimethylsiloxane) Sylgard 184 was poured over themaster structure. See FIG. 1. The PDMS was prepared by mixing 10 partsPDMS to 1 part curing agent. Bubbles were removed by centrifugation at3000 rpm for 30 seconds. The PDMS mixture was poured into a mold placedover the surface of the silicon or glass wafer. The PDMS mixture wascured into a flexible solid at 75° C. for four hours. The cured solidstructure was peeled away from the rigid master structure.

An adhesion-enhancing thin film was applied to a glass substrate(Corning 1737 glass). The thin film was applied by dipping the glasssubstrate in hexamethyldisilane (HMDS) and drying with N₂. SU-8 epoxy(Summers Optical, Ft. Washington, Pa.) was applied to the substrate andthe PDMS mold was pressed into the epoxy. A roller was used to press themold into the epoxy to generate a uniformly thin layer of epoxy betweenthe glass substrate and the PDMS mold. See FIG. 2. The epoxy was curedXenon ultraviolet lamp for 95 seconds. The PDMS mold was peeled awayfrom the glass substrate. A high refractive index dielectric thin filmwas deposited over the epoxy structure. See FIG. 3.

The colorimetric resonant reflection biosensor structure fabricated asdescribed above was illuminated with collimated white light and theexpected resonant reflection peak spectra (FIG. 4) were obtained in air,water, and glycerol. The peak wavelength value (PWV) shift between waterand glycerol (refractive index change of Δn=0.1424) is 18.87 nm,indicating a shift coefficient of ΔPWV/Δn=138. This sensor sensitivityis consistent with colorimetric resonant reflection biosensor structuresproduced by other methods.

1. A method for fabrication of a colorimetric resonant reflection biosensor structure comprising: (a) applying a liquid or semi-solidmaterial that is capable of being transformed or cured into a flexiblesolid over a rigid master structure; (b) transforming the liquid orsemi-solid material into a flexible master structure, wherein theflexible master structure has the rigid master structure embossed into afirst surface of the flexible master structure; (c) peeling the flexiblemaster structure from the rigid master structure; (d) placing the firstsurface of the flexible master structure onto a liquid or semi-solidlayer, wherein the liquid or semi-solid layer is on a rigid substrate;(e) transforming or curing the liquid or semi-solid layer into a solidlayer; (f) peeling the flexible master structure from the solid layer;and (g) applying a high refractive index dielectric film or reflectivematerial over the solid layer, whereby a colorimetric resonantreflection biosensor structure is fabricated.
 2. The method of claim 1,further comprising the step of immobilizing one or more specific bindingsubstances to the high refractive index film or reflective material,whereby a colorimetric resonant reflection biosensor is fabricated. 3.The method of claim 1, wherein the rigid master structure is etched intothe surface of a silicon or glass wafer.
 4. The method of claim 1,wherein the liquid or semi-solid layer is epoxy, a polymer, a cement, asolvent free radiation addition polymerizable crosslinkable material ora resin.
 5. The method of claim 4, wherein the crosslinkable material isan acrylate epoxy urethane based material.
 6. The method of claim 1,wherein the liquid or semi-solid material is polydimethylsiloxane. 7.The method of claim 1, wherein an adhesion-enhancing thin film is placedbetween the liquid or semi-solid layer and the rigid substrate beforethe first surface of the flexible master structure is placed on theliquid or semi-solid layer.
 8. The method of claim 1, wherein the rigidsubstrate is glass, plastic, polymer or epoxy.
 9. The method of claim 1,wherein the liquid or semi-solid layer is transformed or cured by anelectron beam, ultraviolet light or heat.
 10. The method of claim 1,wherein the flexible master structure comprises a grating patternselected from the group consisting of squares, triangles, sinusoidalwaves, inverted “u” shapes, lines, circles, ellipses, trapezoids, ovals,rectangles, hexagons, phase-quantized terraced surface relief patternswhose groove pattern resembles a stepped pyramid, and concentric rings.11. The method of claim 1, wherein the flexible master structurecomprises a grating pattern having a periodic spacing of between about0.1 microns to about 2.0 microns.
 12. The method of claim 1, wherein theflexible master structure comprises a submicron grating pattern.
 13. Themethod of claim 12, wherein the grating pattern has a periodic spacingof between about 0.2 microns to about 0.6 microns.